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22 July 2019

Total Bile Acids: The Value of Fifth Generation Tests

Bile acids are water-soluble, amphipathic end products of cholesterol metabolism and are involved in liver, biliary and intestinal diseases. They are formed in the liver and are absorbed in the small intestine before being excreted. The fundamental role of bile acids is to aid in the digestion and absorption of fats and fat-soluble vitamins in the small intestine.¹

Intrahepatic Cholestasis of Pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder. It can be indicated by pruritus, jaundice, elevated total bile acids and/or serum transaminases and usually affects women during the second and third trimester of pregnancy.^2,3

Intrahepatic Cholestasis of pregnancy or Obstetric Cholestasis is a condition that restricts the flow of bile through the gallbladder resulting in a build-up of bile acids in the liver.³ Due to the build-up, bile acids leak into the bloodstream where they are detected at concerning levels. It is an extremely serious complication of pregnancy that can lead to the increased risk of premature birth or even stillbirth, as such it is vital that women with the disease are monitored carefully.

In healthy pregnancies, there is very little increase in total bile acid levels although a slight increase is likely to be seen in the third trimester. Measurement of total bile acids in serum is thought to be the most suitable method of diagnosing and monitoring ICP.⁶

According to several reports total bile acid levels in ICP can reach as high as 100 times the upper limit of a normal pregnancy. It has been reported that a doubling in maternal serum bile acids, results in a 200% increased risk of stillbirth with total bile acids thought to trigger the onset of preterm labour. Additionally, bile acids can affect the foetal cardiovascular system as it has been found that there are often cardiac rhythm disturbances in the foetus due to the elevated bile acids in circulation.⁵

Although it is a rare condition, with only 0.3-0.5% of women likely to develop ICP, it can have extreme risks and so it is important to properly diagnose and monitor the condition.⁶ ICP increases the risk of meconium staining of the amniotic fluid and is reported to be a sign of foetal distress. This complication is found in 16-58% of all ICP cases, worryingly 100% of cases have resulted in foetal death. The frequency of this condition is found to be greater in pregnancies with higher levels of serum total bile acids.

Did you know?

Liver disease is the only major cause of death still increasing every year with 2 million deaths per year being caused by it.4

Risk factors

There are several risk factors associated with ICP such as a family history of ICP, use of oral contraceptives, assisted reproduction techniques and multiple gestation. Genetic influence accounts for approximately 15% of ICP cases. Dietary selenium is a contributing environmental factor as serum selenium levels often decrease throughout pregnancy. Further to this, incidences of ICP rise in the winter months, most likely due to the fact selenium levels are naturally less during these months.^7,8

Total Bile Acids

In addition to ICP, bile acid levels are also measured in the diagnosis of other liver disorders. The bile acids test in an extremely sensitive indicator of liver function, capable of detecting changes in hepatic function before clinical symptoms arise, thus providing valuable information that standard liver function tests cannot. As a result of its high sensitivity, bile acids can be used to assess liver function in transplant patients, allowing monitoring of the transplant success and of antirejection therapy. The bile acids test is most beneficial when used in conjunction with standard liver function tests such as ALT and AST which are markers of liver damage rather than liver function.

Measurement of Total Bile Acids

The enzyme cycling method, also known as theFifth Generation Bile Acids test, is a method that allows for signal amplification through cycled regeneration reactions as can be seen in Figure 1. In the presence of Thio-NAD, the enzyme 3-α hydroxysteroid dehydrogenase (3-α HSD) converts bile acids to 3-keto steroids and Thio-NADH. The reaction is reversible and 3-α HSD can convert 3-keto steroids and Thio-NADH to bile acids and Thio-NAD. In the presence of excess NADH, the enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 405 nm and is proportional to the amount of total bile acids in the sample. The analysing capability of the fifth generation total bile acids assay is far beyond the performance of conventional bile acid tests.^10,11

Figure 1: The assay principle⁹

Inadequacies of Traditional Bile Acids Assays

Determining the cause and extent of liver damage is important in guiding treatment decisions and preventing disease progression. Standard liver function tests include; ALT, AST, ALP, GGT and Bilirubin. The measurement of TBA is most beneficial in conjunction with these standard liver tests and offers unrivalled sensitivity allowing identification of early stage liver dysfunction. There are several commercial methods available for the detection and measurement of TBA in serum. Traditional TBA tests based on the enzymatic method use nitrotetrazolium blue (NBT) to form a formazan dye. The reaction is measured at 546nm and the intensity of the colour is proportional to the concentration of bile acids.

Newer methods such as the enzyme cycling method or fifth generation methods offer many advantages including greater sensitivity, liquid reagents, small sample volumes and reduced instrument contamination from formazan dye. Additionally, the fifth generation assay does not suffer from interference from lipaemic or haemolytic samples. Both lipemia and haemolysis are common in new-borns and pregnant women, so this further supports that the fifth generation test is more sensitive for these sample types.¹²

Find out more about the Randox Total Bile Acids Assay

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Total Bile Acids Whitepaper

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References
[1] The continuing importance of bile acids in liver and intestinal disease. A.f., Hofmann. 1999, Arch Intern Med, pp. 2647-2658.
[2] Diagnostic and Therapeutic Profiles of Serum Bile Acids in Women with Intrahepatic Cholestasis of Pregnancy – A Pseudo-Targeted Metabolomics Study. Cui, Yue. Xu, Biao. Zhang, Xiaoqing. He, Yifan. Shao, Yong. Ding, Min. s.l. : Clinica Chimica, 2018, Vol. 483.
[3] Randox Laboratories. Bile Acids Test for Obstetric Cholestasis – A serious complication of pregnancy. 2012.
[4] British Liver Trust (2019) Facts about Liver Disease, Available at: https://www.britishlivertrust.org.uk/about-us/media-centre/facts-about-liver-disease/ (Accessed: 18th June 2019).
[5] .Geenes, Victoria. Williamson, Catherine. 17, s.l. : World J Gastroenterol, 2009, Vol. 15.
[6] Howland, Genevieve. Cholestasis of Pregnancy: Why You Can’t Ditch the Itch. Mama Natural. [Online] December 22, 2018. [Cited: February 19, 2019.] https://www.mamanatural.com/cholestasis-of-pregnancy/.
[7] Bile Acid Levels and Risk of Adverse Perinatal Outcomes in Intrahepatic Cholestasis of Pregnancy: A Meta-Analysis. Cui, Donghua, et al.
[8] Intrahepatic Cholestasis of Pregnancy. Chivers, Sian. Williamson, Catherine. 7, 2018, Vol. 28.
[9] Masoud, N; Neill, S.H. Serum bile acids as a sensitive biological marker for evaluating hepatic effects of organic solvents. Available from URL: https://www.ncbi.nlm.nih.gov/pubmed/23885947 [Accessed 1 November 2018]
[10] Microassay of Serum Bile Acids by an Enzymatic Cycling Method. Komiyama, Y, et al. 10, s.l. : Chemical and Pharmaceutical Bulletin, 1982, Vol. 30.
[11] Evaluation of a Colorimetric Enzymatic Procedure for Determining the Total Bile Acids in the Blood. Agape, V, et al. 3, s.l. : Minerva Gastroenterologica e Dietologica, 1989, Vol. 35.
[12] Total Bile Acids Test & Clinical Diagnosis. Diazyme. 2019.

#LoveYourLiver this January. This month, we are taking a closer look at Liver Cirrhosis.

Liver cirrhosis occurs when the healthy tissue of the liver is replaced with scar tissue (fibrosis) due to long-term liver damage. Liver cirrhosis can result in liver failure which can be fatal.

Liver complications such as liver disease and cirrhosis can be detrimental if it is not treated or monitored. Liver disease is the only major cause of death still increasing year-on-year. Globally, deaths due to liver cirrhosis have increased from 676,000 in 1980 to over 1 million in 2010 (NCBI, 2014). Cirrhosis and other chronic liver diseases have increased by 12.4% from 2006-2016 and was the cause of 1,256,900 deaths in 2016 (Global Burden of Disease, 2016).

There are a few factors that increase the risk of liver cirrhosis. The three main factors are heavy alcohol consumption, an undiagnosed hepatitis infection, particularly hepatitis C, and non-alcoholic steatohepatitis (a more severe form of non-alcoholic fatty liver disease) due to obesity.

There are numerous symptoms associated with liver cirrhosis. Some of the more severe symptoms include:

Jaundice – yellowing of the skin and whites of the eyes
Personality changes, confusion, difficulty concentrating, memory loss, or hallucinations
A tendency to bleed or bruise easily
In women, abnormal periods
In men, enlarged breasts, a swollen scrotum (the loose sac of skin that contains the testicles) or shrunken testicles
Vomiting
Diarrhea
Stomach pain – swollen or bloated stomach

Liver cirrhosis cannot be cured, but the aim of treatment is to manage the symptoms and complications, and to stop the condition getting worse.

#LoveYourLiver and prevent or reduce the symptoms of liver cirrhosis through: moderating alcohol consumption, not sharing needles to inject drugs, using a condom during sex, taking medications as prescribed, and maintaining a healthy weight.

The early stages of liver cirrhosis usually does not present any symptoms and is often first detected using routine blood tests. Liver cirrhosis can be diagnosed and monitored through the following routine blood tests:

Alanine Aminotransferase (ALT)

ALT is one of the enzymes within the aminotransferases group and are among the most sensitive liver enzymes. The normal concentration levels of ALT in the blood are low, however, when the liver is damaged, such as liver cirrhosis, the levels of ALT increase. During the diagnosis of liver cirrhosis, the root cause of the damage can be established, such as disease, drug or injury. ALT is commonly measured alongside AST as part of the hepatic panel.

Aspartate Aminotransferase (AST)

AST is an enzyme found throughout the body. Elevated concentration levels of AST in the blood is directly correlated to the severity of the tissue damage. AST also allows for the root cause of the damage to be diagnosed. Excessive levels are indicative of damage due to acetaminophen overdose or acute viral hepatitis. Moderately high levels are indicative of alcohol abuse. Slightly high levels are indicative of cirrhosis.

AST is commonly measured alongside ALT as part of the hepatic panel, although ALT levels are higher in most types of liver damage.

Albumin

Albumin is a special protein made in the liver and provides the body with the proteins it requires to grow and repair tissue. The body requires a proper balance of albumin to prevent fluid from seeping out of blood vessels. Decreased concentrations levels of this protein in the blood is an indicator of liver cirrhosis.

Randox supply a range of third party clinical diagnostic hepatic reagents to aid in the diagnosis and managing the complications of liver cirrhosis. All reagents are available for use on a range of third party biochemistry analysers. Randox offer the following hepatic reagents to diagnose liver cirrhosis:

Alanine Aminotransferase (ALT)

Aspartate Aminotransferase (AST)

Albumin

Randox also offer the following high performance and unique tests to diagnose liver cirrhosis:

5^th Generation Bile Acids

Vanadate Oxidation Bilirubin

Why choose Randox reagents?

Randox offers the largest range of chemistries
Liquid ready-to-use reagents available
Automated applications for a wide range of clinical analysers
Excellent correlation to reference methods
Wide measuring ranges
Flexible pack sizes
Official accreditation to national and international standards including UKAS, ISO 13485:2003, and FDA.
Easy fit reagents
Easy read reagents

To request an application for your specific analyser, contact reagents@randox.com

For more information on liver function or to view our hepatic panel, visit https://www.randox.com/liverfunction/